喜树碱对HaCaT细胞低氧诱导因子1α表达的影响

Effects of camptothecin on the expression of hypoxia-inducible factor-1α in HaCaT cells

目的 观察喜树碱对低氧（2％O2）培养下HaCaT细胞低氧诱导因子1 α（HIF-1α）表达的影响,探讨外用喜树碱治疗银屑病可能的作用机制.方法 将HaCaT细胞分为实验组和溶媒组（对照组）,实验组为喜树碱＋ DMEM培养液,终浓度分别为12.5、25、50、100、200 nmol/L;溶媒组为含二甲基亚砜（DMSO）的DMEM培养液.不同浓度的喜树碱作用于HaCaT细胞12、24、48、72 h,CCK8法检测细胞增殖;用以上浓度处理低氧诱导12h的HaCaT细胞,Western印迹检测细胞HIF-1α蛋白的表达.将HaCaT细胞分为常氧组和低氧组,每组内再分喜树碱干预组（100 nmol/L）和非干预组（溶媒对照组）,培养12h后实时荧光定量PCR检测HIF-1α mRNA的相对表达.统计分析采用SPSS16.0软件进行Levene＇s test、单因素方差分析、Dunnett-t检验和析因分析.结果 不同浓度喜树碱作用12、24、48、72 h后对HaCaT细胞的增殖有抑制作用,呈浓度和时间依赖关系;200 nmol/L喜树碱作用12h,100、200 nmol/L喜树碱作用24 h细胞增殖抑制率分别为（17.66±6.46）％、（33.11±4.63）％、（56.31±1.69）％,与同时段溶媒对照组间差异有统计学意义（均P＜0.05）.低氧诱导12h,25、50、100、200 nmol/L喜树碱处理后HIF-1α蛋白表达分别为0.348±0.065、0.261±0.112、0.115±0.043、0.045±0.024,与溶媒组（1.445±0.329）比较,差异有统计学意义（均P＜0.05）.喜树碱干预（100 nmol/L）与非干预的HIF-1αmRNA表达（△Ct值）分别为：常氧组-5.575±0.29、-5.451±0.21;低氧组-6.543±0.57、-6.203±0.31;低氧较常氧条件下HaCaT细胞HIF-1α mRNA的表达下调,差异有统计学意义（F=29.856,P＜ 0.05）;喜树碱干预和非干预组间HaCaT细胞HIF-1α mRNA的表达在常氧和低氧条件下差异无统计学意义（F=1.667,P＞ 0.05）.结论 100 nmol/L喜树碱可抑制HaCaT细胞HIF-1α蛋白表达,对HIF-1αmRNA的表达水平无明显影响.

Objective To estimate the effects of camptothecin （CPT） on the expression of hypoxia-inducible factor-1α （HIF-1α） in HaCaT cells under hypoxic conditions （2% O2）,and to explore the potential therapeutic mechanism of topical CPT for psoriasis.Methods Some HaCaT cells were classified into 6 groups：5 test groups cultured in Dulbecco＆#39;s modified Eagle＆#39;s medium （DMEM） with the presence of CPT at 12.5,25,50,100 and 200 nmol/L respectively,and 1 control group cultured in DMEM with the presence of dimethyl sulfoxide （DMSO）.All the 6 groups of cells were cultured under normoxic conditions for 12,24,48 or 72 hours or under hypoxic conditions for 12 hours.Cell counting kit-8 （CCK-8） assay was conducted to estimate the proliferation of HaCaT cells after the normoxic culture,and Western blot to quantify the protein expression of HIF-1α after the hypoxic culture.Some HaCaT cells were classified into a normoxia group （21% O2） and a hypoxia group （2% O2）,and each group was divided into a CPT （100 nmol/L）-treated subgroup and a non-intervention subgroup （treated with the vehicle）.After 12-hour culture,real-time fluorescencebased quantitative PCR was performed to measure the mRNA expression of HIF-1α.Statistical analysis was carried out by using Levene＆#39;.s test,one-way analysis of variance,Dunnett-t test and factorial analysis with the SPSS16.0 software.Results After treatment with CPT at 12.5-200 nmol/L for 12-72 hours,the proliferation of HaCaT cells was inhibited in a concentration-and time-dependent manner.More concretely,the cell proliferation rates were inhibited by 17.66% ± 6.46%,33.11% ± 4.63% and 56.31% ± 1.69% respectively in HaCaT cells after 12-hour treatment with 200 nmol/L CPT as well as 24-hour treatment with 100 and 200 nmol/L CPT compared with the control group at the corresponding time points （all P 〈 0.05）.The protein expression level of HIF-1 α was significantly decreased in HaCaT cells after 12-hour treatment with CPT at 12.5,25,50,100 and 200 nmol/L under hypoxic conditions compared with the control group （0.348 ± 0.065,0.261 ± 0.112,0.115 ± 0.043,0.045 ± 0.024 vs.1.445 ± 0.329,all P〈 0.05）.The mRNA expression level of HIF-1α （expressed as △Ct） in the CPT-treated subgroup and non-intervention subgroup was-5.575 ± 0.29 and -5.451 ± 0.21 respectively in the normoxia group,significantly higher than that in the hypoxia group （-6.543 ± 0.57 and -6.203 ± 0.31 respectively,F =29.856,P 〈 0.05）,while there was no significant difference between the CPT-treated and non-intervention subgroups （F =1.667,P 〉 0.05）.Conclusions CPT at 100 nmol/L could inhibit the expression of HIF-1α protein,but had no obvious effect on that of HIF-1α mRNA.